Optimization of the Medium and Cultivation Conditions of Penicillium roquefortii f39 Producing the Diketopiperazine Alkaloid Roquefortine

We optimized the medium for cultivation of Penicillium roquefortii f39, a producer of roquefortine. In this medium, the roquefortine yield increased 1.5–2-fold. An increase in roquefortine content was associated with high biomass yield, but not with an increase in biosynthetic activity of the mycelium. Direct correlation was found between extracellular roquefortine concentration and the amount of the inoculum. The use of sucrose in the inoculum medium allowed us to increase the concentration of roquefortine during fermentation to 90 mg/l.     

Factors contributing to roquefortine yield variability during cultivation of penicillium roquefortii

Variability in the roquefortine yield was shown to be associated with its consumption by the mycelium during isolation of the end product, which depended on temperature, time of culture liquid storage, and biomass concentration. This was also related to the presence in chloroform of chlorocarbonic acid ethyl ester that reacted with roquefortine.     

Factors Contributing to Roquefortine Yield Variability during Cultivation of Penicillium roquefortii

Variability in the roquefortine yield was shown to be associated with its consumption by the mycelium during isolation of the end product, which depended on temperature, time of culture liquid storage, and biomass concentration. This was also related to the presence in chloroform of chlorocarbonic acid ethyl ester that reacted with roquefortine.     

Study of conditions of production of roquefortine and other metabolites of Penicillin roqueforti.

Experiments to determine optimum yields of roquefortine, isofumigaclavine A, and PR toxin, metabolites from Penicillum roqueforti Thom, were performed. Four strains, isolated from blue cheese, and five liquid media were evaluated, although not all permutations were studied. Sucrose (15%)-yeast extract (2%) was the medium chosen for time-course studies at 25 and 15 degrees C using one favorable strain. At 25 degrees C, maximum estimated yields of roquefortine were about 100 mg/liter in the mycelium by 16 days, and no subsequent degradation of this alkaloid was observed. On the other hand, production of PR toxin in the medium peaked at 770 mg/liter at 21 days. At 15 degrees C, yields of roquefortine and PR toxin after 49 days were 60 to 70% of the maximum yields obtained at 25 degrees C. However, about three times more isofumigaclavine A (up to 11 mg/liter) was formed in the mycelium at 15 degrees C than at 25 degrees C. All four strains of P. roqueforti procedure both roquefortine and PR toxin on the sucrose-yeast extract medium at 25 degrees C; isofumigaclavine A was detected in all but one strain grown on this medium.     

Regulation aspects of roquefortine production by free and Ca-alginate immobilized mycelia of Penicillium roqueforti

Summary Roquefortine synthesis with free and Ca-alginate immobilized Penicillium roqueforti cells was investigated under different culture conditions. Decreasing Ca-alginate concentration was related to increasing roquefortine production; free cells gave the best results. Formation of roquefortine was three times higher with mannitol and succinate than with sucrose as the carbon source; phosphate inhibited its biosynthesis in free cells by 23% to 32%. Relationships between cell density, ¹⁴C-tryptophan content of cells and roquefortine synthesis were shown. The special morphology of immobilized mycelia was demonstrated.     

Isolation of Penicillium DIERCKX 1901--a producer of diketopiperazine alkaloids rokefortin and 3,12-dihydrokefortin--from permafrost

Secondary metabolites of the three strains of Penicillium aurantiogriseum, isolated from permafrost sediments, were identified. It was found that these fungi synthesized diketopiperazine alkaloids roquefortine and 3,12-dihydroroquefortine. The strain VKM FW-766 synthesized alkaloids in in the course of certain growth-related processes. When the strain was grown on a mineral medium, time courses of roquefortine and 3,12-dihydroroquefortine concentrations were characterized by biphasic curves.     

Penicillium aurantiogriseum Dierckx 1901: Producer of Diketopiperazine Alkaloids (Roquefortine and 3,12-Dihydroroquefortine), Isolated from Permafrost

Secondary metabolites of three strains of Penicillium aurantiogriseumisolated from permafrost sediments were identified. It was found that these fungi synthesized the diketopiperazine alkaloids roquefortine and 3,12-dihydroroquefortine. The strain VKM FW-766 synthesized alkaloids in the course of certain growth-related processes. When the strain was grown on a mineral medium, the time courses of the roquefortine and 3,12-dihydroroquefortine concentrations were characterized by biphasic curves.     

Intracellular and extracellular alkaloids of Penicillium roqueforti

The alkaloid composition of mycelium and culture liquid filtrate of the fungus Penicillium roqueforti IBPM-F-141 was studied. The new metabolite--3,12-dihydroroquefortine, a derivative of roquefortine, the main component of the alkaloid fraction of this culture, has been isolated for the first time. The structure of 3,12-dihydroroquefortine was determined by chemical and physico-chemical methods. In addition to roquefortine and 3,12-dihydroroquefortine, representatives of a new alkaloid group, the clavine alkaloids, e. g. isofumigaclavine A, isofumigaclavine B and festuclavine, were also isolated and identified. The data on the content of these compounds in mycelium and culture medium are presented.     

Formation of alkaloids from Penicillium species fungi during growth on wheat kernels

The ability to produce alkaloids has been studied in 13 strains belonging to 10 species of the genus Penicillium. Most of these strains produce identical ranges of alkaloids when grown on wheat grain and synthetic Abe medium. They are roquefortine, 3,12-dihydroroquefortine, and glandicolines A and B in strain P. chrysogenum VKM F-1987; fumigaclavines A and B, festuclavine, and pyroclasine in P. commune VKM F-308, F-3491, and KBP4; agroclavine 1 and epoxyagroclavine 1 in P. fellutanum VKM F-1073; fellutanine A in P. fellutanum F-3020; roquefortine, 3,12-dihydroroquefortine, meleagrin, and glandicolines A and B in P. glandicola VKM F-743; aurantioclavine in P. nalgiovense VKM F-229; isofumigaclavines A and B, festuclavine, roquefortine, and 3,12-dihydroroquefortine in P. roquefortii VKM F-2389; roquefortine, 3,12-dihydroroquefortine, and meleagrin in P. vitale VKM F-3624; roquefortine and oxaline in P. vulpinum VKM F-256; and alpha-cyclopiazonic acid and rugulovasine B in P. viridicatum C-47. No alkaloids were found in P. rugulosum VKM F-352 grown on wheat grain. A simple method is proposed for isolating alkaloids from affected grains.     

Production of Alkaloids by Fungi of the Genus PenicilliumGrown on Wheat Grain

The ability to produce alkaloids has been studied in 13 strains belonging to ten species of the genus Penicillium. Most of these strains produce identical ranges of alkaloids when grown on wheat grain and synthetic Abe's medium. These are roquefortine, 3,12-dihydroroquefortine, and glandicolines A and B in strain P. chrysogenum VKM F-1987; fumigaclavines A and B, festuclavine, and pyroclavine in P. commune VKM F-308, F-3491, and KBP4; agroclavine 1 and epoxyagroclavine 1 in P. fellutanum VKM F-1073; fellutanine A in P. fellutanum F-3020; roquefortine, 3,12-dihydroroquefortine, meleagrin, and glandicolines A and B in P. glandicola VKM F-743; aurantioclavine in P. nalgiovense VKM F-229; isofumigaclavines A and B, festuclavine, roquefortine, and 3,12-dihydroroquefortine in P. roquefortii VKM F-2389; roquefortine, 3,12-dihydroroquefortine, and meleagrin in P. vitale VKM F-3624; roquefortine and oxaline in P. vulpinum VKM F-256; and -cyclopiazonic acid and rugulovasine B in P. viridicatum C-47. No alkaloids were found in P. rugulosum VKM F-352 grown on wheat grain. A simple method is proposed for isolating alkaloids from affected grain.     

Liquid chromatography method for the quantitation of the breast cancer resistance protein ABCG2 inhibitor fumitremorgin C and its chemical analogues in mouse plasma and tissues

Fumitremorgin C (FTC) was recently discovered to be a potent and selective inhibitor of the breast cancer resistance protein (BCRP/ABCG2). FTC was shown to reverse multidrug resistance mediated by BCRP and to increase the cytotoxicity of several anticancer agents in vitro. To support in vivo studies a reverse phase HPLC method with ultraviolet detection was developed to quantitate FTC in mouse plasma and tissues. Further, assay method validation was performed for the determination of FTC in mouse plasma. Plasma standard curves ranged from 0.03 to 30 microg/ml, while the various tissue assay ranges differed to some extent. The sample preparation consisted of acetonitrile precipitation with separation accomplished with a C18 Novapak column and a C18 pre-column utilizing an isocratic mobile phase of ammonium acetate and acetonitrile. UV detection was set at 225 nm for FTC and at 312 nm for roquefortine, the internal standard. The retention times were approximately 9.5 min for FTC and 13.0 min for roquefortine. The recoveries for FTC and roquefortine from plasma were 90.8+/-5.8% and 111.6+/-13.6, respectively. The reported assay can be used for future study of BCRP resistance in vivo in different biological matrices. Further, we found that a more potent analogue of FTC, Ko143, was able to be extracted and detected, with a maximal UV absorbance at 320 nm under the conditions reported.     

A Branched Biosynthetic Pathway Is Involved in Production of Roquefortine and Related Compounds in Penicillium chrysogenum

Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus Penicillium chrysogenum and derived deletion strains were used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of this pathway were identified as histidyltryptophanyldiketopiperazine (HTD), dehydrohistidyltryptophanyldi-ketopiperazine (DHTD), roquefortine D, roquefortine C, glandicoline A, glandicoline B and meleagrin. Specific genes could be assigned to each enzymatic reaction step. The nonribosomal peptide synthetase RoqA accepts L-histidine and L-tryptophan as substrates leading to the production of the diketopiperazine HTD. DHTD, previously suggested to be a degradation product of roquefortine C, was found to be derived from HTD involving the cytochrome P450 oxidoreductase RoqR. The dimethylallyltryptophan synthetase RoqD prenylates both HTD and DHTD yielding directly the products roquefortine D and roquefortine C without the synthesis of a previously suggested intermediate and the involvement of RoqM. This leads to a branch in the otherwise linear pathway. Roquefortine C is subsequently converted into glandicoline B with glandicoline A as intermediates, involving two monooxygenases (RoqM and RoqO) which were mixed up in an earlier attempt to elucidate the biosynthetic pathway. Eventually, meleagrin is produced from glandicoline B involving a methyltransferase (RoqN). It is concluded that roquefortine C and meleagrin are derived from a branched biosynthetic pathway.     

Modified arabinogalactans from callus culture <Emphasis Type="Italic">Silene vulgaris</Emphasis> (M.) G.

The cultivation of Silene vulgaris (M.) G. callus culture on the nutrient mediums contained carbohydrates, phytohormones, nitrogen, and phosphate has led to the modification of the arabinogalactan structure from the cell walls. It was noticed that a sucrose concentration increase in the cultivation medium led to an increase of the arabinogalactan fragment yield with a molecular weight more than 300 kDa and a decrease of the yield of fragments with molecular weight less than 300 kDa. The sucrose concentration increase in the nutrient medium entailed the increase of arabinose and galactose content in the fragment with the molecular weight more than 300 kDa and a decrease in the fragment with a molecular weight of 100–300 kDa. On the nutrient medium containing a mix of sucrose and arabinose, the yield of the fraction with a molecular weight more than 300 kDa and the amount of arabinose residues increased, and the yield of minor fragments and the content of arabinose and galactose residues, included in these, decreased. On the medium containing an increased concentration of 2,4-dichlorphenoxyacetic acid, the yield of high-molecular fragment and the content of arabinose residues are two times increased. The decreasing of the amount of arabinose and galactose residues in the fragment with a molecular weight more than 300 kDa was observed at a lack of nitrogen or phosphate in the nutrient medium.     

Effect of calcium chloride on the growth and ethanol production by free cells of Zymomonas mobilis

Summary The effect of calcium chloride concentration on the growth rate and ethanol production using free cells of Zymomonas mobilis was studied. There was no appreciable change in rates of cell mass production and ethanol formation in the medium containing upto 2g/L CaCl₂. On further increase in CaCl₂ concentration, the rates started decreasing. However, the ethanol yield decreased and biomass yield increased with increase in CaCl₂ concentration.     

Influence of Selenium on the Production of T-2 Toxin by <Emphasis Type="Italic">Fusarium poae</Emphasis>

The objective of this study was to investigate the effects of selenium on the production of T-2 toxin by a Fusarium poae strain cultured in a synthetic medium containing different concentrations of selenium. The T-2 toxin contents in fermentative products were evaluated by a high performance liquid chromatography (HPLC). The results showed that the production of T-2 toxin was correlated with the concentration of selenium added to the medium. In all three treatments, the addition of 1 mg/L selenium to the medium resulted in a lower toxin yield than the control (0 mg/L); the yield of the toxin began to increase when selenium concentration was 10 mg/L, while it decreased again at 20 mg/L. In summary, T-2 toxin yield in the fermentative product was affected by the addition of selenium to the medium, and a selenium concentration of 20 mg/L produced the maximum inhibitory effect of T-2 toxin yield in the fermentative product of F. poae.     

Macronutrients requirements of the dinoflagellate Protoceratium reticulatum

The basal L1 medium was found to be unsatisfactory for culturing the red tide dinoflagellate Protoceratium reticulatum at a high growth rate and biomass yield. The L1 medium enhanced with phosphate to a total concentration of 217 μM supported the highest attainable growth rate and biomass yield. Once the phosphate concentration exceeded 6× L1, phosphate inhibited the dinoflagellate growth and negatively affected cell viability. At the optimal phosphate concentration of 217 μM, an increase in nitrate concentration over the range of 882–8824 μM, did not affect cell growth and yield. Nitrate did not inhibit growth at any of the concentrations used. Clearly, the basal nitrate level in L1 is sufficient for effectively culturing P. reticulatum. At the ranges of phosphate and nitrate concentrations tested, cell volume was not sensitive to the concentration of nutrients but the concentration of phosphate affected both the specific cell number and cell volume growth rates. Elevated levels of nutrients supported their intracellular accumulation. Cell-specific production of yessotoxin was not influenced by concentration of phosphate in the culture medium, but elevated (>1764 μM) nitrate concentration did enhance the yessotoxin level. Phosphate concentration that maximized biomass yield also maximized volumetric production of yessotoxin in the culture broth.     

不同发酵条件下产甘油假丝酵母有机酸代谢的研究

产甘油假丝酵母 (Candidaglycerolgenesis)发酵产生的有机酸对丙三醇产品质量和产率均有影响。发现在发酵其它条件恒定 ,装液比和玉米浆浓度增加时 ,发酵液总酸是递增的。在装液比为 0 2和玉米浆浓度为 8g L时 ,丙酮酸和乳酸在细胞生长期可分别积累达 4 1g L和 1 0g L ,比正常发酵时增加 2倍以上 ,丙三醇产率也低 ;然而 ,装液比为 0 0 8和玉米浆浓度为 4g L时 ,丙酮酸和乳酸产生较低 ,丙三醇产率较高 ,但乙酸积累比供氧不足时高 ,可达 2 6g L。发酵过程中有机酸被细胞代谢 ,含量逐渐下降 ,如在初糖浓度为 1 0 0g L时 ,有机酸在细胞生长期积累至高峰后 ,丙三醇和有机酸随之均降低至较低含量 ,并且丙酮酸或乳酸可以转化为乙酸。此外 ,在外加一些添加剂时对其产生有机酸也有影响 ,如添加 1 %油酸和VB1时可以降低乙酸的积累 ,同时增加丙酮酸的含量 ,丙三醇产量也有所增加 ;而丙酮酸结构类似物氟代丙酮酸和亚硫酸盐促进乙酸的产生 ,使酮戊二酸合成减少 ,丙三醇产量约增加 2 0 %。     

Effect of magnesium concentration in the culture medium on the trace element requirement of yeasts]

The effect of different magnesium concentrations in the culture liquid (ranging from 2=6 to 220 mg/l) on the accumulation of Mg and Mn ions in the yeast Candida guilliermondii was studied during their continuous cultivation on purified liquid paraffins. A reverse correlation between the accumulation of magnesium and manganese in the yeast biomass was established. The magnesium content in the biomass increased with an increase of its concentration in the nutrient medium. The biomass yield was optimal at a concentration of magnesium ions in the nutrient medium of 10=25 mg/l. Under these conditions the content of magnesium ions was 0.4 mg % and that of manganese ions (upon their concentration in the nutrient medium of 1=2 mg/l) was 20 mg%.     

Novel Key Metabolites Reveal Further Branching of the Roquefortine/Meleagrin Biosynthetic Pathway

Metabolic profiling and structural elucidation of novel secondary metabolites obtained from derived deletion strains of the filamentous fungus Penicillium chrysogenum were used to reassign various previously ascribed synthetase genes of the roquefortine/meleagrin pathway to their corresponding products. Next to the structural characterization of roquefortine F and neoxaline, which are for the first time reported for P. chrysogenum, we identified the novel metabolite roquefortine L, including its degradation products, harboring remarkable chemical structures. Their biosynthesis is discussed, questioning the exclusive role of glandicoline A as key intermediate in the pathway. The results reveal that further enzymes of this pathway are rather unspecific and catalyze more than one reaction, leading to excessive branching in the pathway with meleagrin and neoxaline as end products of two branches.     

Formation of secreted acid phosphatase during the growth of Saccharomyces cerevisiae yeasts on different sources of carbon and nitrogen nutrition

The effect of certain components in the growth medium on the secretion of acid phosphatase was studied with Saccharomyces cerevisiae. The presence of phosphate at a concentration of 10 mM in the medium inhibited the formation of repressible forms of this enzyme. The synthesis of the secreted enzyme depended on the sources of carbon and nitrogen nutrition. The enzyme yield was highest in a medium with sucrose as a carbon source and ammonium chloride as a nitrogen source. The secretion of acid phosphatase is stimulated by an increase in the sugar content and a deficiency of the nitrogen source in the medium.